Use of amino acid sequences or their corresponding nucleic acids derived from Mycobacterium tuberculosis for the diagnosis and prevention of tuberculosis infection, and diagnostic kits and vaccines derived therefrom

Abstract

To provide an in vitro testing method for detecting Mycobacterium tuberculosis infection in a subject.SOLUTION: A method disclosed herein involves the use of at least six peptides derived from Mycobacterium tuberculosis, the at least six peptides comprising at least one T-cell epitope in association with ESAT6, CFP10 and, optionally, TB7.7 and being selected from a group consisting of TAWITAVVPGLMV (SEQ ID NO:24), ELMARAAVLGSAH (SEQ ID NO:21), RPVRRVLLFVVPSSGPAP (SEQ ID NO:70), GSVRQLPSVLKPPLITLRTLTLSG (SEQ ID NO:71), GEIIFISGRLNGaa (SEQ ID NO:13), AVIVRSELLTQYL (SEQ ID NO:22), LAWITAWPGLMV (SEQ ID NO:85), GEIIFISGRLNG (SEQ ID NO:86), or SALLRRLSTCPPES (SEQ ID NO:87).SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to the use of amino acid sequences or their corresponding nucleic acids derived from Mycobacterium tuberculosis (Mycobacterium tuberculosis) for the diagnosis and prevention of tuberculosis infection, as well as diagnostic kits and vaccines derived therefrom.

[0002] More specifically, the present invention refers to the use of gene sequences or parts thereof, characterized in that they belong to a class of genes that are induced, repressed or conserved in vitro and ex vivo in human macrophages currently infected with Mycobacterium tuberculosis, as well as corresponding peptides or conserved peptides or proteins, for the preparation of specific biomarkers for the diagnosis and prevention of active or latent disease.

[0003] Laboratory diagnosis of M. tuberculosis infection and the development of active disease is crucial to ensure the specificity, rapidity, and effectiveness of treatment.

[0004] Current diagnostic procedures for active tuberculosis disease are based on microscopy, culture, or molecular methods. A positive microscopic result requires a high concentration of mycobacteria in the biological sample (5–10,000 cells / mL), and the sensitivity of these tests is typically below 60% due to their low specificity and inability to distinguish between MTB (Mycobacterium tuberculosis) and nontuberculous mycobacteria.

[0005] Culture testing does not guarantee that clinical reporting will occur within an acceptable timeframe. In fact, MTB colonies are not visible until 10–24 days after plating on solid or liquid media due to the slow growth of the bacteria in vitro. (1) Furthermore, although culture testing is considered highly sensitive, it produces false-negative results at a rate of approximately 10–30% and is expensive.

[0006] Molecular biological tests are highly sensitive and specific, comparable in sensitivity to culture tests, and can rapidly differentiate MTB from nontuberculous mycobacteria. However, their sensitivity is significantly lower when samples contain low concentrations of mycobacteria, i.e., when microscopy is negative. They are available only in highly specialized laboratories and are very expensive.

[0007] Tuberculosis remains a global public health emergency, with high-incidence countries lacking the economic resources needed for effective diagnosis and planned treatment programs, and low-incidence industrialized countries struggling to diagnose infected population segments.

[0008] Currently, in high-incidence countries, effective diagnosis of TB (tuberculosis) is based on microscopic examination of MTB (sensitivity 40-90%) and culture testing (sensitivity 70-90%), which requires a waiting time of 2-6 weeks for culture. In industrialized countries with low incidence, effective diagnosis of TB is based on microscopy and culture testing, while differentiation of non-tuberculous MTB from mycobacteria or diagnosis of microscopy-negative samples is based on molecular testing (sensitivity 70-90%).

[0009] The traditional diagnostic test for diagnosing latent or inactive M. tuberculosis infection is the tuberculin skin test, an economical, rapid in vivo diagnostic test. This test has been standardized for approximately 50 years and can accurately and rapidly detect infection, enabling essential epidemiological surveillance of the incidence and prevalence of M. tuberculosis infection. From a public health perspective, the tuberculin skin test has enabled surveillance of infection incidence and prevalence to achieve global disease control. From a preventive and clinical perspective, it has enabled identification of infection through contact with active TB carriers and the establishment of therapeutic defenses against M. tuberculosis infection aimed at preventing the onset of new cases. Therefore, diagnosing latent infection is a fundamental element of the fight against TB in both high- and low-incidence countries.

[0010] Various cellular and molecular immunological studies have revealed that in vitro contact with MTB or its antigens induces a strong cellular response characterized by high production of interferon-γ (IFN-γ). This suggests that the identification of lymphocytes releasing IFN-γ in response to mycobacteria or their antigens, or the measurement of the cytokine itself, may be an equivalent method to the tuberculin skin test for diagnosing established infection.

[0011] Regarding this issue, two new diagnostic kits for M. tuberculosis infection, QuantiFERON-TB Gold and T-SPOT TB, have recently become commercially available. These kits utilize proteins or peptides derived from the MTB gene located in the RD1 differentiation region of the MTB genome to stimulate IFN-γ production in circulating T lymphocytes. The cost of these kits is prohibitive for widespread use in countries with high tuberculosis incidence. The sensitivity of the two commercially available kits is comparable, with sensitivity and specificity ranging from 70% to 90% and 80% to 95%, respectively (2). Furthermore, the sensitivity and specificity of these kits used in high-incidence areas compared with the traditional tuberculin skin test remain controversial. Indeed, there are cases in which skin tests have proven more sensitive than QuantiFERON (3), and in other cases in which the two tests are comparable (4).

[0012] The main limitations of the tuberculin skin test, still widely used today using either the multiple prick method or the Mantoux intradermal method, are its complex procedure and poor specificity. The test involves two separate visits for tuberculin administration and test reading, each performed by a specialized healthcare professional. For healthcare professionals, contamination of the intradermal syringe poses a risk to the healthcare professional in cases of TB and HIV co-infection. Regarding the specificity of the tuberculin skin test, purified protein derivative (PPD) is known to cross-react with Mycobacterium bovis Bacillus Calmette-Guerin (BCG), which is used in antituberculous vaccines, and various environmental nontuberculous mycobacteria that share high sequence similarity with the genome of Bacillus koch (5, 6). Therefore, subjects who have been vaccinated with BCG or who have recently been exposed to a virulent strain of MTB, such as the laboratory-designated stock strain H37Rv (ATCC 27294), will have a positive tuberculin test, even if the reaction is weaker than that of MTB-infected subjects. Therefore, positivity criteria have been established to distinguish between MTB reactions and those resulting from anti-M. bovis BCG or nontuberculous mycobacterial immunization.

[0013] Such criteria, however, are not sufficiently specific to diagnose M. tuberculosis infection in populations that have been vaccinated or have had long-term exposure to environmental mycobacteria.

[0014] Regarding the prevention of MTB infection, the only vaccine currently available for the prevention of Mycobacterium tuberculosis disease is M. bovis Bacillus Calmette-Guérin (BCG) (ATCC27291), a vaccine based on an attenuated Mycobacterium bovis strain that has been in use worldwide for approximately 75 years.

[0015] In view of the above, it is clear that there is a need to provide new diagnostic kits and vaccines based on the use of specific peptides suitable to overcome the drawbacks of previous techniques.

[0016] There have been several studies on the response of peripheral blood lymphocytes to MTB proteins and peptides in the presence of latent infection or recent contact with TB patients, and in active M. tuberculosis disease, using the ELISPOT assay to detect blood mononuclear cells suitable for producing IFN-γ upon stimulation (7-15). ELISPOT is a technique that allows the detection of the frequency of T lymphocytes that produce cytokines in response to stimulation with one or more antigens (which can be proteins, peptides, or other target molecules) by stimulating mononuclear blood cells in culture plates with sensitized antibodies against cytokines (e.g., IFN-γ).

[0017] T lymphocytes recognize antigens via their T cell antigen receptors (TCRs) when presented in the form of peptides (8–12 amino acids long) that represent epitopes and are bound to molecules of the major histocompatibility complex (MHC), a family of receptors expressed on the plasma membrane of all nucleated cells (for MHC class I molecules) and antigen-presenting cells, such as dendritic cells and macrophages (for MHC class II molecules). In humans, the MHC system is represented by various isotype variants: HLA-A, HLA-B, and HLA-C for class I molecules, and HLA-DP, HLA-DQ, and HLA-DR for class II molecules. Each of these molecules exhibits a different number of allelic variants. Because the antigen repertoire recognized by a T cell TCR is related to the ability of the target antigen-presenting cell MHC receptor to bind peptides generated by digestion of the antigen protein, an essential condition for recognizing peptides as antigens and subsequently activating antigen-specific T lymphocytes is their susceptibility to binding to the MHC receptor.

[0018] The genes encoding HLA molecules are among the most frequently polymorphic genes in the human genome. In this context, it is noteworthy that many of the differences between the individual allelic products of these molecules result from base mutations that code for amino acid sequence alterations in the regions involved in antigen peptide binding. These sequence variations determine the binding properties of each allelic variant of an HLA molecule, allowing the antigen peptide repertoire and the allelic polymorphism to form a trimolecular complex with the TCR of T lymphocytes, resulting in the activation of these lymphocytes.

[0019] It should be pointed out that in the recognition of MTB protein antigens, each mycobacterial epitope can bind to one or more HLA allelic variants, but not all allelic variants need to be expressed in the population. Furthermore, since each non-homozygous subject expresses at least two allelic variants of HLA-A, HLA-B, HLA-C, HLA-DP, and HLA-DQ, as well as two to four HLA-DR variants, different epitopes may be recognized in the context of the same or different allelic variants of different isotypes in many subjects, including the population under investigation.

[0020] Therefore, it is clear that the set of peptides suitable for binding to the various allelic variants of HLA isotypes expressed by each individual in a population may differ. This raises the need to utilize a set of antigenic peptides suitable for reproducing, as comprehensively as possible, the MTB peptide epitope repertoire recognized by T lymphocytes from individuals within the population under investigation. In this context, it should be noted that although the entire MTB genome sequence has been decoded and is available for research, the antigens used to date in immunological tests for the diagnosis of M. tuberculosis infection have been derived from studies on the biochemical characterization of the mycobacterium during in vitro growth in culture medium, rather than from ex vivo experiments.

[0021] U.S. Patent Application No. 2006 / 0115847 discloses an immunological diagnostic method for M. tuberculosis infection based on a combination of epitopes derived from proteins encoded in the genome region of M. tuberculosis that do not occur in the BCG vaccine or the most common nontuberculous mycobacteria. While the experimental section of the patent application reports results for various tested proteins, the distribution of patient responses to single peptides is not entirely uniform. In fact, as shown in Figure 1, some TB patients are peptide-insensitive. Regarding the patient response rates to selected peptides, Table 6 shows that the individually tested peptide CFP10 induced a response in 10 out of 15 patients, i.e., a response frequency of 66.66%. Furthermore, by utilizing a combination of multiple peptides, sensitivity results were obtained for 92% of patients with latent TB (i.e., PPD+), 88% of patients with active TB, and 90% of patients receiving antibiotic treatment, as shown in Table 7 of the patent. Summary of the Invention

[0022] In a previous study, the present authors identified a group of genes preferentially transcribed by MTB-infected human macrophages and characterized these genes as belonging to the deletion region of the M. bovis-BCG vaccine strain (WO2005 / 021790).

[0023] The present authors analyzed MTB-expressed proteins in human macrophages, both in in vitro primary cultures and ex vivo in bronchoalveolar lavage (BAL) samples from patients with active pulmonary TB. Using software developed by the applicants that allows for the analysis of class II histocompatibility molecule binding to peptides from the entire MTB genome, several proteins were selected that proved to be significantly effective from an immunological point of view. In summary, this study compared M. tuberculosis gene expression in three different growth media: synthetic medium (Sauton's), monocyte-derived human macrophages (MDMs) infected in vitro with M. tuberculosis, and alveolar macrophages (AMs) from bronchoalveolar lavage (BAL) samples from patients with pulmonary TB but before antibiotic treatment.

[0024] From the nine gene clusters thus obtained, 100 proteins were initially selected according to combination criteria (regulation of expression, immunogenicity, M. tuberculosis complex specificity, etc.) From these 100 proteins, a further 30 protein clusters were selected, which gave positive immunological reactions in whole blood of TB patients (see Table 1).

[0025] After subsequent selection from four groups of subjects, namely, pulmonary TB before antibiotic treatment (n=13), healthy individuals with recent exposure (blood relatives of TB patients) PPD+ (n=8), healthy individuals with long-term exposure to TB patients (occupational exposure of healthcare workers) PPD+ (n=5), and BCG-vaccinated negative controls, PPD- (n=4), 43 peptides were initially designed, synthesized, and tested.

[0026] Next, the six most sensitive and specific peptides were selected (see Table 2), and the test was repeated using an expanded sample set (see Tables 3 to 5 and Figures 1 to 7).

[0027] The results obtained using the above six peptides and a peptide belonging to ESAT6, a highly immunogenic protein included in both of the above-mentioned commercial kits (Fig. 8), were compared.

[0028] In summary, all selected peptides elicited T cell responses. In particular, peptide number 3 (SEQ ID NO: 71 of the present invention) showed sensitivity comparable to, and possibly even greater than, the control peptide derived from the ESAT-6 protein, which has multiple epitopes. It is noteworthy that, based on the panel evaluated (i.e., by evaluating all post-hoc data for each individual peptide), the six multi-epitope peptides were recognized by approximately 75% of subjects with active TB (in this series, comparable to QuantiFERON-TB Gold in a tube, as shown in Table 4). The data are fully consistent with the results obtained in a subgroup of patients tested simultaneously with the six peptides in the same wells (shown in Table 5).

[0029] Optimal diagnostic sensitivity of peptides is related to optimal specificity. Indeed, reported peptide responses have been limited to active TB subjects, recent exposed contacts, and exposed healthcare workers (Table 4 and Figure 9). Although the data must be supported by a larger number of subjects, it is noteworthy that no peptide responses were detected in a control subject vaccinated with M. bovis BCG or in three subjects who tested positive for QuantiFERON-TB Gold in Tube.

[0030] Furthermore, these peptides can increase the sensitivity of commercially available tests, the current gold standard for diagnosing M. tuberculosis infection (Figure 10). In subjects with active TB, the six selected peptides can increase QuontiFERON-TB Gold-in-Tube sensitivity from 75% to 89% (+14%) and from 71% to 83% (+13%), both when assessed in a panel and when tested directly in the same well (see Tables 4 and 5).

[0031] Therefore, a specific object of the present invention is the use of at least six peptides derived from Mycobacterium tuberculosis and comprising ESAT6 and CFP10, and optionally at least one T cell epitope related to TB7.7, as biomarkers in an in vitro test for the detection of a Mycobacterium tuberculosis infection in a subject, wherein said peptides are selected from the group consisting of TAWITAVVPGLMV (SEQ ID NO: 24), ELMARAAVLGSAH (SEQ ID NO: 21), RPVRRVLLFVVPSSGPAP (SEQ ID NO: 70), GSVRQLPSVLKPPLITLRTLTLSG (SEQ ID NO: 71), GEIIFISGRLNGaa (SEQ ID NO: 13), AVIVRSELLTQYL (SEQ ID NO: 22), LAWITAVVPGLMV (SEQ ID NO: 85), GEIIFISGRLNG (SEQ ID NO: 86) or SALLRRLSTCPPES (SEQ ID NO: 87). According to an embodiment of the present invention, the peptides are the following six peptides: ELMARAAVLGSAH (SEQ ID NO: 21), RPVRRVLLFVVPSSGPAP (SEQ ID NO: 70), GSVRQLPSVLKPPLITLRTLTLSG (SEQ ID NO: 71), AVIVRSELLTQYL (SEQ ID NO: 22), TAWITAVVPGLMV (SEQ ID NO: 24), and GEIIFISGRLNGaa (SEQ ID NO: 13). According to a further embodiment of the present invention, the peptides are the following six peptides: ELMARAAVLGSAH (SEQ ID NO: 21), RPVRRVLLFVVPSSGPAP (SEQ ID NO: 70), GSVRQLPSVLKPPLITLRTLTLSG (SEQ ID NO: 71), AVIVRSELLTQYL (SEQ ID NO: 22), LAWITAVVPGLMV (SEQ ID NO: 85), and GEIIFISGRLNG (SEQ ID NO: 86).According to another embodiment of the present invention, the peptides are all of the following nine peptides: TAWITAVVPGLMV (SEQ ID NO: 24), ELMARAAVLGSAH (SEQ ID NO: 21), RPVRRVLLFVVPSSGPAP (SEQ ID NO: 70), GSVRQLPSVLKPPLITLRTLTLSG (SEQ ID NO: 71), GEIIFISGRLNGaa (SEQ ID NO: 13), AVIVRSELLTQYL (SEQ ID NO: 22), LAWITAVVPGLMV (SEQ ID NO: 85), GEIIFISGRLNG (SEQ ID NO: 86) and SALLRRLSTCPPES (SEQ ID NO: 87).

[0032] The present invention also relates to the use of at least one peptide derived from Mycobacterium tuberculosis and comprising ESAT6 and CFP10, and optionally at least one T cell epitope related to TB7.7, as a biomarker in an in vitro test for the detection of a Mycobacterium tuberculosis infection in a subject, wherein said peptide is selected from the group consisting of TAWITAVVPGLMV (SEQ ID NO: 24), ELMARAAVLGSAH (SEQ ID NO: 21), RPVRRVLLFVVPSSGPAP (SEQ ID NO: 70), GSVRQLPSVLKPPLITLRTLTLSG (SEQ ID NO: 71), GEIIFISGRLNGaa (SEQ ID NO: 13), AVIVRSELLTQYL (SEQ ID NO: 22), LAWITAVVPGLMV (SEQ ID NO: 85), GEIIFISGRLNG (SEQ ID NO: 86) or SALLRRLSTCPPES (SEQ ID NO: 87). In particular, the peptide may be LAWITAVVPGLMV (SEQ ID NO: 85) or TAWITAVVPGLMV (SEQ ID NO: 24).

[0033] A further object of the present invention is an in vitro method for diagnosing infection with Mycobacterium tuberculosis in a subject, said method comprising incubating a blood sample comprising lymphocytes from said subject in the presence of at least six peptides derived from Mycobacterium tuberculosis and comprising ESAT6 and CFP10, and optionally at least one T-cell epitope related to TB7.7, for a time and under conditions sufficient to stimulate said lymphocytes to produce effector molecules, wherein the presence or level of said effector molecules indicates that the subject is infected with a Mycobacterium species or is infected with a Mycobacterium species. and wherein the at least six peptides are selected from the group consisting of TAWITAVVPGLMV (SEQ ID NO: 24), ELMARAAVLGSAH (SEQ ID NO: 21), RPVRRVLLFVVPSSGPAP (SEQ ID NO: 70), GSVRQLPSVLKPPLITLRTLTLSG (SEQ ID NO: 71), GEIIFISGRLNGaa (SEQ ID NO: 13), AVIVRSELLTQYL (SEQ ID NO: 22), LAWITAVVPGLMV (SEQ ID NO: 85), GEIIFISGRLNG (SEQ ID NO: 86), or SALLRRLSTCPPES (SEQ ID NO: 87).

[0034] According to one embodiment of the method of the present invention, the peptides are the following six peptides: ELMARAAVLGSAH (SEQ ID NO: 21), RPVRRVLLFVVPSSGPAP (SEQ ID NO: 70), GSVRQLPSVLKPPLITLRTLTLSG (SEQ ID NO: 71), AVIVRSELLTQYL (SEQ ID NO: 22), TAWITAVVPGLMV (SEQ ID NO: 24), and GEIIFISGRLNGaa (SEQ ID NO: 13). According to a further embodiment of the method of the present invention, the peptides are the following six peptides: ELMARAAVLGSAH (SEQ ID NO: 21), RPVRRVLLFVVPSSGPAP (SEQ ID NO: 70), GSVRQLPSVLKPPLITLRTLTLSG (SEQ ID NO: 71), AVIVRSELLTQYL (SEQ ID NO: 22), LAWITAVVPGLMV (SEQ ID NO: 85), and GEIIFISGRLNG (SEQ ID NO: 86). According to another embodiment, the peptides are all nine of the following peptides: TAWITAVVPGLMV (SEQ ID NO: 24), ELMARAAVLGSAH (SEQ ID NO: 21), RPVRRVLLFVVPSSGPAP (SEQ ID NO: 70), GSVRQLPSVLKPPLITLRTLTLSG (SEQ ID NO: 71), GEIIFISGRLNGaa (SEQ ID NO: 13), AVIVRSELLTQYL (SEQ ID NO: 22), LAWITAVVPGLMV (SEQ ID NO: 85), GEIIFISGRLNG (SEQ ID NO: 86) and SALLRRLSTCPPES (SEQ ID NO: 87).

[0035] The object of the present invention is to provide an in vitro method for diagnosing an infection with Mycobacterium tuberculosis in a subject, comprising: The method comprises: subjecting a blood sample comprising lymphocytes from the subject in the presence of at least one peptide derived from Mycobacterium tuberculosis and comprising ESAT6 and CFP10, and optionally at least one T cell epitope associated with TB7.7; incubating for a time and under conditions sufficient to stimulate said lymphocytes to produce effector molecules; the presence or level of said effector molecule is indicative of lymphocytes derived from a subject infected with or previously exposed to Mycobacterium species; The at least one peptide is selected from the group consisting of TAWITAVVPGLMV (SEQ ID NO:24), ELMARAAVLGSAH (SEQ ID NO:21), RPVRRVLLFVVPSSGPAP (SEQ ID NO:70), GSVRQLPSVLKPPLITLRTLTLSG (SEQ ID NO:71), GEIIFISGRLNGaa (SEQ ID NO:13), AVIVRSELLTQYL (SEQ ID NO:22), LAWITAVVPGLMV (SEQ ID NO:85), GEIIFISGRLNG (SEQ ID NO:86), or SALLRRLSTCPPES (SEQ ID NO:87). For example, the peptide can be LAWITAVVPGLMV (SEQ ID NO:85) or TAWITAVVPGLMV (SEQ ID NO:24).

[0036] A further object of the present invention is a method for the in vitro diagnosis of infection with Mycobacterium tuberculosis in a subject, comprising: The method comprises incubating a blood sample containing lymphocytes from the subject with ESAT6 and CFP10, and optionally TB7.7, and measuring the release of interferon gamma by the lymphocytes; the method is characterized in that the incubation is carried out in the presence of at least six peptides selected from the group consisting of TAWITAVVPGLMV (SEQ ID NO: 24), ELMARAAVLGSAH (SEQ ID NO: 21), RPVRRVLLFVVPSSGPAP (SEQ ID NO: 70), GSVRQLPSVLKPPLITLRTLTLSG (SEQ ID NO: 71), GEIIFISGRLNGaa (SEQ ID NO: 13), AVIVRSELLTQYL (SEQ ID NO: 22), LAWITAVVPGLMV (SEQ ID NO: 85), GEIIFISGRLNG (SEQ ID NO: 86) or SALLRRLSTCPPES (SEQ ID NO: 87); The method provides a method for detecting Mycobacterium tuberculosis with a higher level of sensitivity and / or selectivity compared to the sensitivity and / or selectivity achieved with ESAT6 and CFP10, and optionally TB7.7. According to an embodiment of the present invention, the peptides are the following six peptides: ELMARAAVLGSAH (SEQ ID NO: 21), RPVRRVLLFVVPSSGPAP (SEQ ID NO: 70), GSVRQLPSVLKPPLITLRTLTLSG (SEQ ID NO: 71), AVIVRSELLTQYL (SEQ ID NO: 22), TAWITAVVPGLMV (SEQ ID NO: 24), and GEIIFISGRLNGaa (SEQ ID NO: 13). According to a further embodiment of the present invention, the peptides are the following six peptides: ELMARAAVLGSAH (SEQ ID NO: 21), RPVRRVLLFVVPSSGPAP (SEQ ID NO: 70), GSVRQLPSVLKPPLITLRTLTLSG (SEQ ID NO: 71), AVIVRSELLTQYL (SEQ ID NO: 22), LAWITAVVPGLMV (SEQ ID NO: 85) and GEIIFISGRLNG (SEQ ID NO: 86). According to another embodiment of the present invention, the peptides are all of the following nine peptides: TAWITAVVPGLMV (SEQ ID NO: 24), ELMARAAVLGSAH (SEQ ID NO: 21), RPVRRVLLFVVPSSGPAP (SEQ ID NO: 70), GSVRQLPSVLKPPLITLRTLTLSG (SEQ ID NO: 71), GEIIFISGRLNGaa (SEQ ID NO: 13), AVIVRSELLTQYL (SEQ ID NO: 22), LAWITAVVPGLMV (SEQ ID NO: 85), GEIIFISGRLNG (SEQ ID NO: 86) and SALLRRLSTCPPES (SEQ ID NO: 87).

[0037] The present invention also provides a method for the in vitro diagnosis of infection with Mycobacterium tuberculosis in a subject, comprising: The method comprises incubating a blood sample containing lymphocytes from the subject with ESAT6 and CFP10, and optionally TB7.7, and measuring the release of interferon gamma by the lymphocytes; the method is characterized in that the incubation is further carried out in the presence of at least one peptide selected from the group consisting of TAWITAVVPGLMV (SEQ ID NO: 24), ELMARAAVLGSAH (SEQ ID NO: 21), RPVRRVLLFVVPSSGPAP (SEQ ID NO: 70), GSVRQLPSVLKPPLITLRTLTLSG (SEQ ID NO: 71), GEIIFISGRLNGaa (SEQ ID NO: 13), AVIVRSELLTQYL (SEQ ID NO: 22), LAWITAVVPGLMV (SEQ ID NO: 85), GEIIFISGRLNG (SEQ ID NO: 86) or SALLRRLSTCPPES (SEQ ID NO: 87); The method has an increased level of sensitivity and / or selectivity for detecting Mycobacterium tuberculosis compared to the sensitivity and / or selectivity achieved with ESAT6 and CFP10, optionally TB7.7. The at least one peptide can be LAWITAVVPGLMV (SEQ ID NO: 85) or TAWITAVVPGLMV (SEQ ID NO: 24).

[0038] A further object of the present invention is to The use of at least one biomarker selected from the list consisting of: in an in vitro test for the detection of a mycobacterial infection in a subject, i.e. a human or non-human animal subject: (i) Rv0023, Rv0182c, Rv0290, Rv0601c, Rv0647c, Rv0724A, Rv0890c, Rv1251c, Rv1398c, Rv1478, Rv1497, Rv1575, and Rv157, which are derived from Mycobacterium species or related organisms and contain at least one T cell epitope. a protein selected from Rv1899c, Rv2137c, Rv2333c, Rv2548, Rv2557, Rv2816c, Rv2990, Rv3094c, Rv3107c, Rv3188, Rv3239c, Rv3296, Rv3425, Rv3446c, Rv3479, Rv3482c, and Rv3780; (ii) a homologue of one of the proteins defined in (i) having an amino acid sequence with at least 80% similarity compared to the protein after optimal alignment; and (iii) A peptide fragment of a protein as defined in (i) or (ii), which fragment contains a T-cell epitope or a chemical analogue thereof.

[0039] The Mycobacterium species include M. tuberculosis, M. bovis, M. bovis BCG, M. africanum, M. canetti, M. caprae, M. microti, M. pinnipedii, M. avium, M. avium paratuberculosis, M. avium silvaticum, M. avium "hominissuis", M. colombiense, M. asiaticum, and M. gourdnae. gordonae, M. gastri, M. kansasii, M. hiberniae, M. nonchromogenicum, M. terrae, M. triviale, M. ulcerans, M. pseudoshottsii, M. shottsii, M. triplex, M. genavense, M. florentinum, M. lentiflavum, M. palustre, M. kubicae, M. parascrophulariaceum parascrofulaceum, M. heidelbergense, M. interjectum, M. simiae, M. branderi, M. cookii, M. celatum, M. bohemicum, M. haemophilum, M. malmoense, M. surugaiszulgai, M. leprae, M. lepraemurium, M. lepromatosis, M. africanum, M. botniense, M. chimaera, M. conspicuum, M. doricum, M. farcinogenes, M. heckeshornense, M. intracellulare, M. lacus, M. marinum, M. monacense, M. montefiorense, M. murare murale, M. nebraskense, M. saskatchewanense, M. scrofulaceum, M. shimoidei, M. tusciae, M. xenopi, M. intermedium, M. abscessus, M. chelonae, M. bolletii, M. fortuitum, M. fortuitum subsp. acetamidolyticum, M. boenickei, M. peregrinum, M. porcinum porcinum, M. senegalense, M. septicum, M. neworleansense, M. houstonense, M. mucogenicum, M. mageritense, M. brisbanense, M. cosmeticum, M.M. parafortuitum, M. austroafricanum, M. diernhoferi, M. hodleri, M. neoaurum, M. frederiksbergense, M. aurum, M. vaccae, M. chitae, M. fallax, M. confluentis, M. flavescens, M. madagascariense, M. phlei, M. smegmatis, M. goodii goodii, M. wolinskyi, M. thermoresistibile, M. gadium, M. komossense, M. obuense, M. sphagni, M. agri, M. aichiense, M. alvei, M. arupense, M. brumae, M. canariasense, M. chubuense, M. conceptionense, M. duvalii, M. elephantis, M. gillum gilvum, M. hassiacum, M. holsaticum, M. immunogenum, M. massiliense, M. moriokaense, M. psychrotolerans, M. pyrenivorans, M. vanbaalenii, M. pulveris, M.The Mycobacterium species may be selected from M. arosiense, M. aubagnense, M. caprae, M. chlorophenolicum, M. fluoroanthenivorans, M. kumamotonense, M. novocastrense, M. parmense, M. phocaicum, M. poriferae, M. rhodesiae, M. seoulense, and M. tokaiense. Preferably, the Mycobacterium species is Mycobacterium tuberculosis.

[0040] According to the above use, the peptide fragment defined in (iii) may comprise or consist of an amino acid sequence selected from TAWITAVVPGLMV (SEQ ID NO: 24), AVIVRSELLTQYL (SEQ ID NO: 22), GSVRQLPSVLKPPLITLRTLTLSG (SEQ ID NO: 71), RPVRRVLLFVVPSSGPAP (SEQ ID NO: 70), GEIIFISGRLNGaa (SEQ ID NO: 13), ELMARAAVLGSAH (SEQ ID NO: 21), LAWITAVVPGLMV (SEQ ID NO: 85), GEIIFISGRLNG (SEQ ID NO: 86), SALLRRLSTCPPES (SEQ ID NO: 87).

[0041] The above uses may further comprise the use of one or more mycobacterial proteins selected from ESAT6, CFP10, TB7.7 and PPD, or peptide fragments thereof or chemical analogues derived therefrom.

[0042] A further object of the present invention is to The use of at least one biomarker selected from the list consisting of: in an in vitro test for the detection of a mycobacterial infection in a subject, i.e. a human or non-human animal subject: (i) Rv0023, Rv0182c, Rv0290, Rv0601c, Rv0647c, Rv0724A, Rv0890c, Rv1251c, Rv1398c, Rv1478, Rv1497, Rv1575, and Rv157, which are derived from Mycobacterium species or related organisms and contain at least one T cell epitope. a protein selected from Rv1899c, Rv2137c, Rv2333c, Rv2548, Rv2557, Rv2816c, Rv2990, Rv3094c, Rv3107c, Rv3188, Rv3239c, Rv3296, Rv3425, Rv3446c, Rv3479, Rv3482c, and Rv3780; (ii) a homologue of one of the proteins defined in (i) having an amino acid sequence with at least 80% similarity compared to the protein after optimal alignment; and (iii) a peptide fragment of a protein defined in (i) or (ii) that has a T-cell epitope or a chemical analog thereof; and (iv) a Mycobacterium-derived protein, or a fragment thereof, or a chemical analog thereof, selected from the list consisting of: (a)ESAT; (b) CFP10; (c) TB7.7; and (d) PPD.

[0043] The Mycobacterium species include M. tuberculosis, M. bovis, M. bovis BCG, M. africanum, M. canetti, M. caprae, M. microti, M. pinnipedii, M. avium, M. avium paratuberculosis, M. avium silvaticum, M. avium "hominissuis", M. colombiense, M. asiaticum, and M. gourdnae. gordonae, M. gastri, M. kansasii, M. hiberniae, M. nonchromogenicum, M. terrae, M. triviale, M. ulcerans, M. pseudoshottsii, M. shottsii, M. triplex, M. genavense, M. florentinum, M. lentiflavum, M. palustre, M. kubicae, M. parascrophulariaceum parascrofulaceum, M. heidelbergense, M. interjectum, M. simiae, M. branderi, M. cookii, M. celatum, M. bohemicum, M. haemophilum, M. malmoense, M. surugaiszulgai, M. leprae, M. lepraemurium, M. lepromatosis, M. africanum, M. botniense, M. chimaera, M. conspicuum, M. doricum, M. farcinogenes, M. heckeshornense, M. intracellulare, M. lacus, M. marinum, M. monacense, M. montefiorense, M. murare murale, M. nebraskense, M. saskatchewanense, M. scrofulaceum, M. shimoidei, M. tusciae, M. xenopi, M. intermedium, M. abscessus, M. chelonae, M. bolletii, M. fortuitum, M. fortuitum subsp. acetamidolyticum, M. boenickei, M. peregrinum, M. porcinum porcinum, M. senegalense, M. septicum, M. neworleansense, M. houstonense, M. mucogenicum, M. mageritense, M. brisbanense, M. cosmeticum, M.M. parafortuitum, M. austroafricanum, M. diernhoferi, M. hodleri, M. neoaurum, M. frederiksbergense, M. aurum, M. vaccae, M. chitae, M. fallax, M. confluentis, M. flavescens, M. madagascariense, M. phlei, M. smegmatis, M. goodii goodii, M. wolinskyi, M. thermoresistibile, M. gadium, M. komossense, M. obuense, M. sphagni, M. agri, M. aichiense, M. alvei, M. arupense, M. brumae, M. canariasense, M. chubuense, M. conceptionense, M. duvalii, M. elephantis, M. gillum gilvum, M. hassiacum, M. holsaticum, M. immunogenum, M. massiliense, M. moriokaense, M. psychrotolerans, M. pyrenivorans, M. vanbaalenii, M. pulveris, M.The Mycobacterium species may be selected from Mycobacterium arosiense, M. aubagnense, M. caprae, M. chlorophenolicum, M. fluoroanthenivorans, M. kumamotonense, M. novocastrense, M. parmense, M. phocaicum, M. poriferae, M. rhodesiae, M. seoulense, and M. tokaiense. Preferably, the Mycobacterium species is Mycobacterium tuberculosis.

[0044] According to the above use, the peptide fragment defined in (iii) may comprise or consist of an amino acid sequence selected from TAWITAVVPGLMV (SEQ ID NO: 24), AVIVRSELLTQYL (SEQ ID NO: 22), GSVRQLPSVLKPPLITLRTLTLSG (SEQ ID NO: 71), RPVRRVLLFVVPSSGPAP (SEQ ID NO: 70), GEIIFISGRLNGaa (SEQ ID NO: 13), ELMARAAVLGSAH (SEQ ID NO: 21), LAWITAVVPGLMV (SEQ ID NO: 85), GEIIFISGRLNG (SEQ ID NO: 86), SALLRRLSTCPPES (SEQ ID NO: 87).

[0045] A further object of the present invention is the use of at least one nucleic acid molecule encoding a biomarker selected from the list consisting of: (i) Rv0023, Rv0182c, Rv0290, Rv0601c, Rv0647c, Rv0724A, Rv0890c, Rv1251c, Rv1398c, Rv1478, Rv1497, Rv1575, and Rv157, which are derived from Mycobacterium species or related organisms and contain at least one T cell epitope. a protein selected from Rv1899c, Rv2137c, Rv2333c, Rv2548, Rv2557, Rv2816c, Rv2990, Rv3094c, Rv3107c, Rv3188, Rv3239c, Rv3296, Rv3425, Rv3446c, Rv3479, Rv3482c, and Rv3780; (ii) a homologue of one of the proteins defined in (i) having an amino acid sequence with at least 80% similarity compared to the protein after optimal alignment; and (iii) A peptide fragment of a protein as defined in (i) or (ii), which fragment contains a T-cell epitope or a chemical analogue thereof.

[0046] The Mycobacterium species are M. tuberculosis, M. bovis, M. bovis BCG, M. africanum, M. canetti, M. caprae, M. microti, M. pinnipedii, M. avium, M. avium paratuberculosis, M. avium silvaticum, M. avium "hominissuis", M. colombiense, M. asiaticum, M. gourdnae, M. gordonae, M. gastri, M. kansasii, M. hiberniae, M. nonchromogenicum, M. terrae, M. triviale, M. ulcerans, M. pseudoshottsii, M. shottsii, M. triplex, M. genavense, M. florentinum, M. lentiflavum, M. palustre, M. kubicae, M. parascrophulariaceum parascrofulaceum, M. heidelbergense, M. interjectum, M. simiae, M. branderi, M. cookii, M. celatum, M. bohemicum, M. haemophilum, M. malmoense, M. surugaiszulgai, M. leprae, M. lepraemurium, M. lepromatosis, M. africanum, M. botniense, M. chimaera, M. conspicuum, M. doricum, M. farcinogenes, M. heckeshornense, M. intracellulare, M. lacus, M. marinum, M. monacense, M. montefiorense, M. murare murale, M. nebraskense, M. saskatchewanense, M. scrofulaceum, M. shimoidei, M. tusciae, M. xenopi, M. intermedium, M. abscessus, M. chelonae, M. bolletii, M. fortuitum, M. fortuitum subsp. acetamidolyticum, M. boenickei, M. peregrinum, M. porcinum porcinum, M. senegalense, M. septicum, M. neworleansense, M. houstonense, M. mucogenicum, M. mageritense, M. brisbanense, M. cosmeticum, M.M. parafortuitum, M. austroafricanum, M. diernhoferi, M. hodleri, M. neoaurum, M. frederiksbergense, M. aurum, M. vaccae, M. chitae, M. fallax, M. confluentis, M. flavescens, M. madagascariense, M. phlei, M. smegmatis, M. goodii goodii, M. wolinskyi, M. thermoresistibile, M. gadium, M. komossense, M. obuense, M. sphagni, M. agri, M. aichiense, M. alvei, M. arupense, M. brumae, M. canariasense, M. chubuense, M. conceptionense, M. duvalii, M. elephantis, M. gillum gilvum, M. hassiacum, M. holsaticum, M. immunogenum, M. massiliense, M. moriokaense, M. psychrotolerans, M. pyrenivorans, M. vanbaalenii, M. pulveris, M.The Mycobacterium species is selected from M. arosiense, M. aubagnense, M. caprae, M. chlorophenolicum, M. fluoroanthenivorans, M. kumamotonense, M. novocastrense, M. parmense, M. phocaicum, M. poriferae, M. rhodesiae, M. seoulense, and M. tokaiense. Preferably, the Mycobacterium species is Mycobacterium tuberculosis.

[0047] According to the above use, the peptide fragment in (iii) may comprise or consist of an amino acid sequence selected from TAWITAVVPGLMV (SEQ ID NO: 24), AVIVRSELLTQYL (SEQ ID NO: 22), GSVRQLPSVLKPPLITLRTLTLSG (SEQ ID NO: 71), RPVRRVLLFVVPSSGPAP (SEQ ID NO: 70), GEIIFISGRLNGaa (SEQ ID NO: 13), ELMARAAVLGSAH (SEQ ID NO: 21), LAWITAVVPGLMV (SEQ ID NO: 85), GEIIFISGRLNG (SEQ ID NO: 86), SALLRRLSTCPPES (SEQ ID NO: 87). The above use may further comprise the use of a nucleic acid molecule encoding a mycobacterial protein or a peptide fragment derived therefrom selected from ESAT6, CFP10, TB7.7 and PPD, or homologs thereof.

[0048] A further object of the present invention is to provide antibodies derived from Mycobacterium species and comprising T-cell epitopes, such as Rv0023, Rv0182c, Rv0290, Rv0601c, Rv0647c, Rv0724A, Rv0890c, Rv1251c, Rv1398c, Rv1478, Rv1497, Rv1575, Rv1578c, Rv1899c, An isolated protein selected from the list consisting of Rv2137c, Rv2333c, Rv2548, Rv2557, Rv2816c, Rv2990, Rv3094c, Rv3107c, Rv3188, Rv3239c, Rv3296, Rv3425, Rv3446c, Rv3479, Rv3482c, and Rv3780. The isolated peptide of the protein of the invention may comprise a T-cell epitope or a chemical analog thereof. The isolated peptide may comprise or consist of an amino acid sequence selected from TAWITAVVPGLMV (SEQ ID NO: 24), AVIVRSELLTQYL (SEQ ID NO: 22), GSVRQLPSVLKPPLITLRTLTLSG (SEQ ID NO: 71), RPVRRVLLFVVPSSGPAP (SEQ ID NO: 70), GEIIFISGRLNGaa (SEQ ID NO: 13), ELMARAAVLGSAH (SEQ ID NO: 21), LAWITAVVPGLMV (SEQ ID NO: 85), GEIIFISGRLNG (SEQ ID NO: 86), SALLRRLSTCPPES (SEQ ID NO: 87).

[0049] A further object of the present invention is an isolated nucleic acid molecule encoding a protein or peptide according to the invention as described above.

[0050] The present invention relates to a vector comprising the above-described nucleic acid molecule, and to an isolated cell comprising said vector.

[0051] A further object of the present invention is a kit comprising a container, said container containing at least one protein or at least one peptide or at least one nucleic acid molecule as described above.

[0052] The present invention provides a method for in vitro diagnosis of infection with Mycobacterium species in a subject, comprising: The method comprises incubating a blood sample containing lymphocytes from the subject in the presence of at least one biomarker selected from the list consisting of: for a time and under conditions sufficient to stimulate the lymphocytes to produce effector molecules; The presence or level of said effector molecule is indicative of lymphocytes derived from a subject infected with or previously exposed to Mycobacterium species, the method comprising: (i) Rv0023, Rv0182c, Rv0290, Rv0601c, Rv0647c, Rv0724A, Rv0890c, Rv1251c, Rv1398c, Rv1478, Rv1497, Rv1575, and Rv157, which are derived from Mycobacterium species or related organisms and contain at least one T cell epitope. a protein selected from Rv1899c, Rv2137c, Rv2333c, Rv2548, Rv2557, Rv2816c, Rv2990, Rv3094c, Rv3107c, Rv3188, Rv3239c, Rv3296, Rv3425, Rv3446c, Rv3479, Rv3482c, and Rv3780; (ii) a homologue of one of the proteins defined in (i) having an amino acid sequence with at least 80% similarity compared to the protein after optimal alignment; and (iii) a peptide fragment of a protein as defined in (i) or (ii), bearing a T-cell epitope or a chemical analogue thereof. The subject may be a human or a non-human animal. The incubation between the blood and the biomarker may occur in a test tube, optionally in the presence of heparin and in the presence of added carbohydrates. According to an embodiment of the present invention, the incubation may further occur in the presence of a mycobacterial protein selected from ESAT6, CFP10, TB7.7 and PPD, or a peptide fragment thereof, or a chemical analogue thereof derived therefrom, or a mixture thereof. The Mycobacterium species may be: M. tuberculosis, M. bovis, M. bovis BCG, M. africanum, M. canetti, M. caprae, M. microti, M. pinnipedii, M. avium, M. Avium paratuberculosis, M. Avium silvaticum, M. Avium "hominissuis", M. colombiense, M. asiaticum, M. gordonae, M. gastri gastri, M. kansasii, M. hiberniae, M. nonchromogenicum, M. terrae, M. triviale, M. ulcerans, M. pseudoshottsii, M. shottsii, M. triplex, M. genavense, M. florentinum, M. lentiflavum, M. palustre, M. kubicae, M. parascrofulaceum, M. heidelbergens heidelbergense, M. interjectum, M. simiae, M. branderi, M. cookii, M. celatum, M. bohemicum, M. haemophilum, M. malmoense, M. szulgai, M. lepraeleprae, M. lepraemurium, M. lepromatosis, M. africanum, M. botniense, M. chimaera, M. conspicuum, M. doricum, M. farcinogenes, M. heckeshornense, M. intracellulare, M. lacus, M. marinum, M. monacense, M. montefiorense, M. murale, M. nebrascens nebraskense, M. saskatchewanense, M. scrofulaceum, M. shimoidei, M. tusciae, M. xenopi, M. intermedium, M. abscessus, M. chelonae, M. bolletii, M. fortuitum, M. fortuitum subsp. acetamidolyticum, M. boenickei, M. peregrinum, M. porcinum, M. senegalensis senegalense, M. septicum, M. neworleansense, M. houstonense, M. mucogenicum, M. mageritense, M. brisbanense, M. cosmeticum, M. parafortuitumparafortuitum, M. austroafricanum, M. diernhoferi, M. hodleri, M. neoaurum, M. frederiksbergense, M. aurum, M. vaccae, M. chitae, M. fallax, M. confluentis, M. flavescens, M. madagascariense, M. phlei, M. smegmatis, M. goodii, M. wolinskii wolinskyi, M. thermoresistibile, M. gadium, M. komossense, M. obuense, M. sphagni, M. agri, M. aichiense, M. alvei, M. arupense, M. brumae, M. canariasense, M. chubuense, M. conceptionense, M. duvalii, M. elephantis, M. gilvum, M. hassiacum hassiacum, M. holsaticum, M. immunogenum, M. massiliense, M. moriokaense, M. psychrotolerans, M. pyrenivorans, M. vanbaalenii, M. pulveris, M. allosiensis, M.The Mycobacterium species is selected from Mycobacterium arosiense, M. aubagnense, M. caprae, M. chlorophenolicum, M. fluoroanthenivorans, M. kumamotonense, M. novocastrense, M. parmense, M. phocaicum, M. poriferae, M. rhodesiae, M. seoulense, and M. tokaiense. Preferably, the Mycobacterium species is Mycobacterium tuberculosis.

[0053] According to the method of the present invention described above, the peptide defined in (iii) comprises or consists of an amino acid sequence selected from TAWITAVVPGLMV (SEQ ID NO: 24), AVIVRSELLTQYL (SEQ ID NO: 22), GSVRQLPSVLKPPLITLRTLTLSG (SEQ ID NO: 71), RPVRRVLLFVVPSSGPAP (SEQ ID NO: 70), GEIIFISGRLNGaa (SEQ ID NO: 13), ELMARAAVLGSAH (SEQ ID NO: 21), LAWITAVVPGLMV (SEQ ID NO: 85), GEIIFISGRLNG (SEQ ID NO: 86), SALLRRLSTCPPES (SEQ ID NO: 87). The effector molecule may be selected from interferon gamma, cytokines, interleukins, and TNF-alpha, preferably interferon gamma.

[0054] A further object of the present invention is an isolated antibody specific for a protein or peptide as defined above.

[0055] The present invention also provides a method for the in vitro diagnosis of infection with Mycobacterium tuberculosis in a subject, comprising: The method comprises incubating a blood sample containing lymphocytes from the subject with one or more of ESAT6, CFP10, TB7.7, and / or PPD, and measuring the release of interferon-γ by the lymphocytes; The method further comprises the step of: (i) Rv0023, Rv0182c, Rv0290, Rv0601c, Rv0647c, Rv0724A, Rv0890c, Rv1251c, Rv1398c, Rv1478, Rv1497, Rv1575, and Rv157, which are derived from Mycobacterium species or related organisms and contain at least one T cell epitope. a protein selected from Rv1899c, Rv2137c, Rv2333c, Rv2548, Rv2557, Rv2816c, Rv2990, Rv3094c, Rv3107c, Rv3188, Rv3239c, Rv3296, Rv3425, Rv3446c, Rv3479, Rv3482c, and Rv3780; (ii) a homologue of one of the proteins defined in i) having an amino acid sequence with at least 80% similarity compared to the protein after optimal alignment; and (iii) a peptide fragment of a protein defined in (i) or (ii) that contains a T-cell epitope or a chemical analog thereof; The method is characterized in that it is performed in the presence of at least one biomarker selected from The method has a higher level of sensitivity and / or selectivity for detecting Mycobacterium tuberculosis compared to the sensitivity and / or selectivity of using ESAT6, CFP10, TB7.7, and / or PPD alone.

[0056] Additionally, the present invention provides a vaccine for the treatment or prevention of infection by Mycobacterium species, comprising: The above vaccines are (i) Rv0023, Rv0182c, Rv0290, Rv0601c, Rv0647c, Rv0724A, Rv0890c, Rv1251c, Rv1398c, Rv1478, Rv1497, Rv1575, and Rv157, which are derived from Mycobacterium species or related organisms and contain at least one T cell epitope. a protein selected from Rv1899c, Rv2137c, Rv2333c, Rv2548, Rv2557, Rv2816c, Rv2990, Rv3094c, Rv3107c, Rv3188, Rv3239c, Rv3296, Rv3425, Rv3446c, Rv3479, Rv3482c, and Rv3780; (ii) a homologue of one of the proteins defined in (i) having an amino acid sequence with at least 80% similarity compared to the protein after optimal alignment; and (iii) a peptide fragment of a protein defined in (i) or (ii) that contains a T-cell epitope or a chemical analog thereof; at least one substance selected from the list consisting of: and one or more pharmaceutically acceptable adjuvants, carriers, excipients, and / or diluents.

[0057] According to the vaccine of the present invention, the Mycobacterium species is selected from the group consisting of M. tuberculosis, M. bovis, M. bovis BCG, M. africanum, M. canetti, M. caprae, M. microti, M. pinnipedii, M. avium, M. avium paratuberculosis, M. avium silvaticum, M. avium "hominissuis", M. colombiense, M. asiaticum, M. gourdnae, M. gordonae, M. gastri, M. kansasii, M. hiberniae, M. nonchromogenicum, M. terrae, M. triviale, M. ulcerans, M. pseudoshottsii, M. shottsii, M. triplex, M. genavense, M. florentinum, M. lentiflavum, M. palustre, M. kubicae, M. parascrophulariaceum parascrofulaceum, M. heidelbergense, M. interjectum, M. simiae, M. branderi, M. cookii, M. celatum, M. bohemicum, M. haemophilum, M. malmoense, M.M. szulgai, M. leprae, M. lepraemurium, M. lepromatosis, M. africanum, M. botniense, M. chimaera, M. conspicuum, M. doricum, M. farcinogenes, M. heckeshornense, M. intracellulare, M. lacus, M. marinum, M. monacense, M. montefiorense, M. murare murale, M. nebraskense, M. saskatchewanense, M. scrofulaceum, M. shimoidei, M. tusciae, M. xenopi, M. intermedium, M. abscessus, M. chelonae, M. bolletii, M. fortuitum, M. fortuitum subsp. acetamidolyticum, M. boenickei, M. peregrinum, M. porcinum porcinum, M. senegalense, M. septicum, M. neworleansense, M. houstonense, M. mucogenicum, M. mageritense, M. brisbanense, M. cosmeticum, M.M. parafortuitum, M. austroafricanum, M. diernhoferi, M. hodleri, M. neoaurum, M. frederiksbergense, M. aurum, M. vaccae, M. chitae, M. fallax, M. confluentis, M. flavescens, M. madagascariense, M. phlei, M. smegmatis, M. goodii goodii, M. wolinskyi, M. thermoresistibile, M. gadium, M. komossense, M. obuense, M. sphagni, M. agri, M. aichiense, M. alvei, M. arupense, M. brumae, M. canariasense, M. chubuense, M. conceptionense, M. duvalii, M. elephantis, M. gillum gilvum, M. hassiacum, M. holsaticum, M. immunogenum, M. massiliense, M. moriokaense, M. psychrotolerans, M. pyrenivorans, M. vanbaalenii, M. pulveris, M.The Mycobacterium species is selected from M. arosiense, M. aubagnense, M. caprae, M. chlorophenolicum, M. fluoroanthenivorans, M. kumamotonense, M. novocastrense, M. parmense, M. phocaicum, M. poriferae, M. rhodesiae, M. seoulense, and M. tokaiense. Preferably, the Mycobacterium species is Mycobacterium tuberculosis.

[0058] The vaccine of the present invention can be used in human or non-human animal subjects. The peptide defined in (iii) can comprise or consist of an amino acid sequence selected from TAWITAVVPGLMV (SEQ ID NO: 24), AVIVRSELLTQYL (SEQ ID NO: 22), GSVRQLPSVLKPPLITLRTLTLSG (SEQ ID NO: 71), RPVRRVLLFVVPSSGPAP (SEQ ID NO: 70), GEIIFISGRLNGaa (SEQ ID NO: 13), ELMARAAVLGSAH (SEQ ID NO: 21), LAWITAVVPGLMV (SEQ ID NO: 85), GEIIFISGRLNG (SEQ ID NO: 86), and SALLRRLSTCPPES (SEQ ID NO: 87). Therefore, the present invention relates to a vaccine as defined above for use in preventing infections caused by Mycobacterium species.

[0059] A further object of the present invention is at least one substance selected from the list consisting of: (i) Rv0023, Rv0182c, Rv0290, Rv0601c, Rv0647c, Rv0724A, Rv0890c, Rv1251c, Rv1398c, Rv1478, Rv1497, Rv1575, and Rv157, which are derived from Mycobacterium species or related organisms and contain at least one T cell epitope. a protein selected from Rv1899c, Rv2137c, Rv2333c, Rv2548, Rv2557, Rv2816c, Rv2990, Rv3094c, Rv3107c, Rv3188, Rv3239c, Rv3296, Rv3425, Rv3446c, Rv3479, Rv3482c, and Rv3780; (ii) a homologue of one of the proteins defined in (i) having an amino acid sequence with at least 80% similarity compared to the protein after optimal alignment; and (iii) A peptide fragment of a protein as defined in (i) or (ii), which fragment contains a T-cell epitope or a chemical analogue thereof.

[0060] The present invention further provides a method for assessing the ability of a subject to mount a cellular immune response in vitro, comprising: The method comprises: contacting a sample containing T lymphocytes sensitized to Mycobacterium species, or an antigen or protein containing a T lymphocyte epitope therefrom, with at least one substance selected from the following for a time and under conditions sufficient to stimulate said lymphocytes to produce effector molecules: The presence or level of said effector molecule is indicative of the ability of said subject to mount a cellular immune response, (i) Rv0023, Rv0182c, Rv0290, Rv0601c, Rv0647c, Rv0724A, Rv0890c, Rv1251c, Rv1398c, Rv1478, Rv1497, Rv1575, and Rv157, which are derived from Mycobacterium species or related organisms and contain at least one T cell epitope. a protein selected from Rv1899c, Rv2137c, Rv2333c, Rv2548, Rv2557, Rv2816c, Rv2990, Rv3094c, Rv3107c, Rv3188, Rv3239c, Rv3296, Rv3425, Rv3446c, Rv3479, Rv3482c, and Rv3780; (ii) a homologue of one of the proteins defined in (i) having an amino acid sequence with at least 80% similarity compared to the protein after optimal alignment; and (iii) A peptide fragment of a protein as defined in (i) or (ii), which fragment contains a T-cell epitope or a chemical analogue thereof.

[0061] The present invention will now be described in a specific but non-limiting manner according to preferred embodiments thereof with particular reference to the accompanying drawings. [Brief explanation of the drawings]

[0062] [Figure 1] Analysis of IFN-γ production as a PPD response comparing four subject populations: a. Primary pulmonary TB patients; b. Healthy contacts, TB-exposed, PPD-positive; c. Healthy controls, occupationally exposed to TB, QuantiFERON-positive; d. Negative controls, QuantiFERON-negative, BCG-vaccinated. [Figure 2] Analysis of IFN-γ production in response to the TAWITAVVPGLMV (SEQ ID NO: 24) peptide from four groups of subjects. [Figure 3] Analysis of IFN-γ production in response to AVIVRSELLTQYL (SEQ ID NO: 22) peptide from four groups of subjects. [Figure 4]Analysis of IFN-γ production in response to the GSVRQLPSVLKPPLITLRTLTLSG (SEQ ID NO: 71) peptide from four groups of subjects. [Figure 5] Analysis of IFN-γ production in response to RPVRRVLLFVVPSSGPAP (SEQ ID NO: 70) peptide from four groups of subjects. [Figure 6] Analysis of IFN-γ production in test samples in response to the GEIIFISGRNGaa (SEQ ID NO: 13) peptide. [Figure 7] Analysis of IFN-γ production in test samples in response to the ELMARAAVLGSAH (SEQ ID NO: 21) peptide. [Figure 8] Analysis of test samples for IFN-γ production in response to ESAT6 (QQWNFAGIEAAASAIQGNVTSIHSL - SEQ ID NO: 84). [Figure 9] Frequency of positive tests using only the six peptides SEQ ID NOs: 24, 21, 71, 70, 13, and 22. [Figure 10] Improved sensitivity of the commercially available QuantiFERON TB-Plus test after addition of peptides SEQ ID NOs: 24, 21, 70, 71, 13, 22. [Figure 11] Combination of peptides 1–6. Compared to the sensitivities of 55%, 33%, and 45.5% obtained with the RD1(ESAT6) multi-epitope peptide for TB+ subjects, PPD+ exposed contacts, and HCWs, respectively, the panel achieved sensitivities of 76.3%, 62%, and 66.7% when peptides 1–6 were combined. DETAILED DESCRIPTION OF THE INVENTION [Example]

[0063] Example 1: Identification of M. tuberculosis-expressed proteins in infected human macrophages from biological samples analyzed in vitro and ex vivo Materials and Methods ELISPOT immunodiagnostic test

[0064] The complete procedure for performing the test requires a 96-well plate (MAIPS45, Millipore, Sunnyvale, CA, USA); primary antibody (monoclonal, M-700A, Pierce-Endogen, Rockford, USA, coated with IFN-γ); biotinylated antibody (M-701B, Pierce-Endogen); streptavidin-HRP (Pierce-Endogen); substrate (AEC staining kit, Sigma); and ready-to-use concentrations of stimuli (peptides, PHA, and other antigens).

[0065] The ELISPOT procedure is carried out according to the following steps. Coating: Treat a 96-well plate with 100 μL per well of primary antibody (5 μg / mL) in sterile phosphate-buffered saline (PBS). Cover the plate and incubate at 4°C for 20 hours, then wash the plate four times with 200 μL per well of sterile PBS. After the final wash, remove excess liquid by flicking the plate on absorbent paper.

[0066] Blocking: To prevent non-specific protein binding, add 200 μL of "blocking solution" (sterile PBS containing 10% fetal calf serum (FCS)) per well, incubate the plate at room temperature for 2 hours, and aspirate the "blocking solution."

[0067] Cell preparation and incubation 1. Isolation of mononuclear cells (PBMCs) from venous blood (7 ml, containing EDTA) by density gradient centrifugation (Ficoll-Hypaque, Pharmacia; Uppsala; Sweden) using a rapid method based on the use of filter tubes (LeucoSep™, ARNIKA, Milan) to separate leukocytes. After two washes with 1x PBS (phosphate-buffered saline), cells were collected at a density of 2x10 per 100 μL. 5 The pellet was resuspended in complete medium [RPMI 1640 containing 25 mM HEPES, 10% (v / v) FCS, 2 mM L-glutamine, and 10 U / mL penicillin / streptomycin] to obtain a single pellet. 2. Add 100 μL of cell suspension and 100 μL of each different stimulus per well. 3. Incubate the plate for 40 hours at 37°C in a 5% CO2 incubator. 4. Remove the cells. 5. Wash the plate 4 times with 200 μL per well of PBS, then 4 times with 200 μL per well of "wash buffer" [PBS / 0.05% Tween 20 (Sigma)]. 6. For the final wash, gently flick the plate on absorbent paper to remove excess liquid.

[0068] Incubation with biotinylated antibody Biotinylated antibodies diluted to a concentration of 1 μg / ml in PBS / 4% bovine serum albumin (fraction V, Sigma) were added in a volume of 100 μl per well. The plates were then incubated for 100 minutes at 37°C in a 5% CO2 incubator, and washed four times with "wash buffer." For the final wash, excess liquid was removed by gently flicking the plate on absorbent paper.

[0069] detection For detection, 100 μl of Streptavidin-HRP diluted 1:1000 in Wash Buffer was added per well. The plate was incubated for 30 minutes at room temperature in the dark and then washed four times with Wash Buffer. After the final wash, excess liquid was removed by gently flicking the plate on absorbent paper. 100 μL of substrate was added per well. In parallel, as a control for the enzyme-substrate reaction, a similarly prepared substrate was incubated with 100 μl of diluted Streptavidin-HRP for several minutes. If the reaction was successful, the substrate would change color from light brown to pink.

[0070] Finally, the plates were incubated for 10–20 min at room temperature in the dark. The substrate was discarded, and the plates were washed with water to remove excess, then air-dried for 20 h.

[0071] ELISA test for the identification of IFN-γ in human and animal whole blood samples stimulated with selected peptides and proteins (CMI test procedure).

[0072] result The authors identified a set of proteins expressed by M. tuberculosis in infected human macrophages from biological samples analyzed both in vitro and ex vivo. They compared M. tuberculosis gene expression in three different environments: synthetic medium (Sauton's), monocyte-derived human macrophages (MDMs) infected with M. tuberculosis in culture, and alveolar macrophages (AMs) from bronchoalveolar lavage (BAL) samples from patients with pulmonary TB but before antibiotic treatment.

[0073] From the nine gene groups thus obtained, 100 proteins were initially selected according to combined criteria (immunogenicity, M. tuberculosis complex specificity, etc.) From these 100 proteins, 30 protein groups were again selected, which showed positive immunological reactions in whole blood immunoassays of TB patients.

[0074] [Table 1] JPEG0007719993000002.jpg244170JPEG0007719993000003.jpg114170Adjustment Legend: A: upregulated in AM versus MDM; B: always expressed in AM and MDM; C: upregulated in MDM relative to AM; D: upregulated by Sauton for MDM and / or AM; E: Upregulated in MDM and / or AM relative to Sauton's.

[0075] After subsequent selection from four groups of subjects: pulmonary TB before antibiotic treatment (n=13); healthy individuals with recent exposure (blood relatives of TB patients) PPD+ (n=8); healthy individuals with long-term exposure to TB patients (occupational exposure of healthcare workers) PPD+ (n=5); and BCG-vaccinated negative controls, PPD- (n=4), 43 peptides were initially designed, synthesized, and tested.

[0076] Next, the six most sensitive and specific peptides were selected (see Table 2), and the test was repeated using an expanded sample of interest (see Tables 3-4 and Figures 1-7).

[0077] Table 2 shows the genes selected from MTB, the peptides selected for the T CD4+ cell assay, and their corresponding identification numbers.

[0078] [Table 2]

[0079] [Table 3]

[0080] [Table 4]

[0081] [Table 5]

[0082] The results obtained using the above six peptides and a peptide belonging to the ESAT6 protein, ie, a highly immunogenic protein available in the two commercially available kits mentioned above, were compared.

[0083] The MTB genes that were observed to be induced both during the course of infection in human macrophages and / or in alveolar macrophage samples from patients with active pulmonary TB are listed below.

[0084] A gene that is constantly expressed during intracellular replication in MDMs and AMs: Rv0724A.

[0085] Genes induced in AM and / or MDM versus Sauton's medium: Rv1251c, Rv1478 and Rv3479.

[0086] The two groups of MTB genes share putative functions involved in survival within human host cells (both primary macrophages from in vitro infected healthy donors and alveolar macrophages from TB patients), which has led to their design as biomarkers for intracellular survival of MTB, and the definition of MTB virulence is based solely on the pathogen's ability to invade, survive, and replicate within host cells.

[0087] Furthermore, the authors of this study designed peptide sequences for several gene groups belonging to the same metabolic category to identify "common" protein sequences within that category. This study is based on the hypothesis that similar functional domains utilized in proteins present in many bacterial species are conserved. To find these conserved motifs, a multiple sequence alignment (PSSM) was generated using PSI-BLAST (Position-Specific Iterative Basic Local Alignment Search Tool, http: / / www.ncbi.nlm.nih.gov / BLAST). Once sequences with high homology to the inserted sequence were identified, suitable proteins could be selected to contribute to the generation of a profile for subsequent databank searches. In this method, the number of sequences contributing to the profile generation varies depending on the position of the different sequences.

[0088] The multiple alignment allows the detection of highly conserved and therefore structurally and functionally important residues, which together constitute the "consensus" sequence or sequences of each MTB protein functional group.

[0089] Therefore, we analyzed proteins (induced or repressed in human macrophages) from metabolic function groups (e.g., regulatory proteins, lipid metabolism-related proteins, etc.) detected as "regulated" by M. tuberculosis during the infection process to search for consensus sequences. Using PSI-BLAST, we obtained multiple alignments of diverse sequences and found the most "common" sequence related to peptide synthesis.

[0090] Peptides derived from selected proteins were synthesized and used to detect and quantify MTB-specific T CD4+ lymphocytes using a detection system for IFN-γ-producing cells that employs both ELISPOT technology and highly sensitive TB diagnostic ELISA assays (i.e., QuontiFERON TB-Plus and QuontiFERON CMI). This approach allows for the quantification of the frequency of T cells producing a given cytokine (e.g., IFN-γ) in response to a specific antigenic stimulus, suggesting that the immune system of the treated subject was able to mount an immune response against the peptide if presented with an infectious agent (MTB) encoding the peptide. A second approach allows for the quantification of the total amount of IFN-γ produced by specific T lymphocytes in response to a selected antigen.

[0091] Although this test does not provide evidence of the peptides' ability to induce protection from MTB infection, the resulting detection of lymphocytes specifically and individually recognizing these peptides in subjects infected with MTB or in subjects with active TB is indicative of their immunogenicity (a necessary minimal characteristic) for vaccines and diagnostic tests to be effective. Furthermore, these peptides, alone or in combination with other mycobacterial antigens, can provide a highly sensitive and specific test for diagnosing TB and improve the sensitivity of commercially available tests, the current gold standard for diagnosing M. tuberculosis (Figure 10). When evaluated as a panel and when tested directly in the same well, the six selected peptides can improve the QuantiFERON TB Gold-in-Tube response in subjects with active TB from 75% to 89% (+14%) and from 71% to 83% (+13%), respectively, without reducing the specificity of the assay.

[0092] References 1. EW Koneman, SD Allen, WM Janda, P. Schreckenberger, WC Winn. Color Atlas and Textbook of Diagnostic Microbiology. 5th ed. Lippincott, 1997 a edizione. Lippincott, 1997). 2. Menzies D, Pai M, Comstock G. Ann Intern Med. 2007 Mar 6;146(5):340-54. Meta-analysis: new tests for the diagnosis of latent tuberculosis infection: areas of uncertainty and recommendations for research. 3. Bellete B, Coberly J, Barnes GL, Ko C, Chaisson RE, Comstock GW, Bishai WR. Journal of Infectious Diseases of America. 2002 34:1449-56. 4. Pai M, Gokhale K, Joshi R, Dogra S, Kalantri S, Mendiratta DK, Narang P, Daley CL, Granich RM, Mazurek GH, Reingold AL, Riley LW, Colford JM Jr. JAMA. 2005 293:2746-55. 5. Lalvani A, Pathan AA, Durkan H, Wilkinson KA, Whelan A, Deeks JJ, Reece WH, Latif M, Pasvol G, Hill AV. Lancet. 2001;357:2017-21. 6. Centers for Disease Control and Prevention. MMWR Recomm Rep. 1997 Sep 5;46(RR-15):1-10. 7. Ulrichs T, Munk ME, Mollenkopf H, et al. Eur J Immunol 1998; 28:3949-3958. 8. Ravn P, Demissie A, Eguale T, et al. J Infectious Diseases of America. 1999;179:637-645. 9. Doherty TM, Demissie A, Olobo J, et al. J Clin Microbiol 2002; 40:704-706. 10. Lalvani A, Pathan AA, Durkan H, et al. Lancet 2001; 357: 2017-2021. 11. Lalvani A, Nagvenkar P, Udwadia Z, et al. J Infectious Diseases of America. 2001;183:469-477. 12. Chapman AL, Munkanta M, Wilkinson KA, et al. AIDS. 2002;16:2285-2293. 13. Lalvani A, Pathan AA, McShane H, et al. Journal of the American Thoracic Care Med 2001; 163:824-828. 14.Pathan AA, Wilkinson KA, Klenerman P, et al. J Immunol 2001; 167:5217-5225. 15. LinksLawn SD, Bangani N, Vogt M, Bekker LG, Badri M, Ntobongwana M, Dockrell HM, Wilkinson RJ, Wood R. Utility of interferon-γ ELISPOT assay responses in highly tuberculosis-exposed patients with advanced HIV infection in South Africa. BMC Infect Dis. 2007 Aug 28;7:99 16. De Groot AS, Bosma A, Chinai N, Frost J, Jesdale BM, Gonzalez MA, Martin W, Saint-Aubin C. Vaccine 2001, 19:4385-4395. 17. Brander C, Goulder PJR. Edited by Korber BTM, Brander C, Haynes B, Koup RA, Kuiken C, Moore J, Walker B, Watkins D. Los Alamos National Laboratory, Los Alamos, New Mexico, 2000; I1-I3.

Claims

1. 1. A method for detecting an infection by Mycobacterium tuberculosis in a subject in vitro, comprising: The method comprises: Incubating a blood sample containing lymphocytes from the subject in the presence of a peptide consisting of the amino acid sequence of SEQ ID NO: 14, 15, 16, 17, 18, 19, or 20 for a time and under conditions sufficient to stimulate the lymphocytes to produce interferon-γ; and measuring the release of interferon gamma by said lymphocytes; The method, wherein the presence or level of interferon gamma is indicative of lymphocytes from a subject infected with or previously exposed to Mycobacterium tuberculosis.

2. 2. The method of claim 1, wherein the incubation between the blood and the peptide consisting of the amino acid sequence of SEQ ID NO: 14, 15, 16, 17, 18, 19, or 20 occurs in a test tube.

3. 3. The method of claim 1 or 2, wherein the incubation further occurs in the presence of heparin.

4. 4. The method of claim 1, wherein the incubation further occurs in the presence of added carbohydrates.

5. 5. The method of any one of claims 1 to 4, wherein said incubation occurs in the presence of a mycobacterial protein further selected from ESAT6, CFP10, TB7.7 and PPD, or a mixture thereof.

6. The method according to any one of claims 1 to 5, wherein the subject is a human.

7. The method according to any one of claims 1 to 5, wherein the subject is a non-human animal.

8. 1. A method for in vitro detection of infection with Mycobacterium tuberculosis in a subject, comprising: The method comprises incubating a blood sample containing lymphocytes from the subject with one or more of ESAT6, CFP10, TB7.7, and / or PPD, and measuring the release of interferon-γ by the lymphocytes; The method is characterized in that the incubation is further carried out in the presence of a peptide consisting of the amino acid sequence of SEQ ID NO: 14, 15, 16, 17, 18, 19, or 20; A method having an increased level of sensitivity and / or selectivity for detecting Mycobacterium tuberculosis compared to the sensitivity and / or selectivity of using ESAT6, CFP10, TB7.7, and / or PPD alone.

9. 1. A method for assessing the ability of a subject to mount a cellular immune response in vitro, comprising: The method comprises: contacting a sample containing T lymphocytes sensitized to Mycobacterium tuberculosis or an antigen or protein containing a T lymphocyte epitope derived therefrom with a peptide consisting of the amino acid sequence of SEQ ID NO: 14, 15, 16, 17, 18, 19, or 20 for a time and under conditions sufficient to stimulate the lymphocytes to produce interferon-γ; measuring the release of interferon gamma by said lymphocytes; The method, wherein the presence or level of interferon gamma is indicative of the subject's ability to mount a cell-mediated immune response.

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